The present invention relates to a composition and to a method for extracting DNA. More specifically, the present invention relates to a composition and to a method to extract DNA from dried biological samples on solid substrates, including but not limited to, buccal smears, semen and especially blood. The method can be conducted in a single-tube. The DNA extracted in accordance with the present invention can be used for DNA amplification reactions, DNA sequencing, DNA restriction analysis and DNA hyridization.
Blood and other biological samples are commonly archived by applying the sample to filter paper and allowing it to dry. These samples are used for newborn screening, diagnostic testing, and felon databasing. Samples are typically applied to filter paper cards and allowed to dry. Two types of filter paper cards are prevalent, Schleicher and Schuell 903 (SandS 903) and Fitzco FTA(trademark) cards. SandS 903 paper is a heavy, highly absorbent cotton bond paper. Fitzco FTA(trademark) cards are similar, but are treated with several compounds (U.S. Pat. No. 5,496,562) designed to kill pathogens and resist bacterial growth and DNA degradation. These compounds include Tris, EDTA, SDS, and uric acid. Fitzco claims that the xe2x80x9cmembranes are disrupted and the DNA explodes out of the nucleus causing high molecular weight DNA to become entangled in the fibers of the paperxe2x80x9d (Fitzco Product Information).
To perform molecular diagnostic studies such as polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), the DNA must be purified in situ or extracted from the paper matrix. The currently available protocols require lengthy enzymatic digestions, incubations, and separation steps. Many of these protocols produce very little, poor quality DNA and are not amenable to high-throughput applications. Furthermore, protocols for FTA paper recommend amplification directly from the paper, since it is reported that it is difficult to get the DNA into solution.
There are several drawbacks to this last approach. There is no method to measure the amount or purity of DNA available in the paper. The protocol for RFLP requires enzymatic digestion to release the DNA from the paper matrix. Since the RFLP protocol involves enzymatic digestion, it poses the problems mentioned above.
Paper punchers are available, but this process is difficult to automate reliably. Static electricity and normal air movements can cause mishandling of the paper punches. Finally, the smallest available punchers are 1 mm in diameter, which does not fit in a 384-well microtiter plate. This places a lower limit on PCR reactions, which can increase the consumption of expensive reagents.
Therefore, it is desirable to develop a simple, rapid, high-throughput method to release the DNA from the paper matrix and a composition to be used in this method. DNA in solution is more easily applied to automated processes.
The present invention relates to a composition and to a method for extracting DNA. More specifically, the present invention relates to a composition and to a method to extract DNA from biological samples, especially blood dried on cellulosic material, such as paper. The method can be conducted in a single-tube. The DNA extracted in accordance with the present invention can be used for DNA amplification reactions, DNA sequencing, DNA restriction analysis and DNA hybridization.
In accordance with one embodiment of the present invention, a DNA extraction composition (solution) is provided which comprises formamide, citrate, a suitable buffer and optionally a non-ionic detergent. In accordance with a second embodiment of the present invention, a method for extracting DNA from biological samples is provided. In one aspect of the invention, the biological sample is blood. In a second aspect of the invention, the blood is dried on a cellulosic material. The method comprises contacting the biological sample with the DNA extraction solution, heating the resultant mixture and isolating supernatant containing the extracted DNA. This method is suitable for extracting DNA in a single tube.
Current procedures for extracting DNA from certain biological samples, such as dried blood on cellulosic material such as cotton based papers (e.g., Schleicher and Schuell 903 (SandS 903) and Fitzco FTA(trademark) cards), require lengthy enzymatic digestions, incubations and separation steps. In addition, compounds used as preservatives in FTA(trademark) paper would become soluble as a result of DNA extraction or DNA amplification reactions and inhibit enzymes.,used for DNA amplification. Thus, it was desired to develop a simple, rapid, high-throughput method to release the DNA from the paper matrix. It was further desired to develop a composition to be used in this method which would not only serve to extract the DNA, but would also serve to remove or inactivate the compounds present in FTA(trademark) paper. The method and composition described herein satisfies these desires and produces DNA that is suitable for use in molecular procedures, including amplification, sequencing, hybridization and restriction analysis.
In accordance with one aspect of the present invention, a composition is provided which is capable of (i) extracting DNA from a biological sample, such as buccal smears, semen and particularly blood, dried on a cellulosic material, such as cotton based papers and (ii) removing or inactivating compounds present in the cellulosic material that may otherwise interfere in analysis of the DNA. The DNA composition of the present invention is further capable of extracting sufficient DNA for molecular analysis in a single tube in a simple method. According to the present invention, the DNA extraction composition comprises (1) formamide, (2) citrate and (3) a buffer. The DNA extraction composition may optionally comprises a non-ionic detergent.
The DNA composition comprises formamide in an amount from about 5% to about 90%, preferably from about 5% to about 50%, and more preferably about 10%. The DNA extraction composition comprises citrate in an amount from about 5 mM to about 60 mM, preferably from about 10 mM to about 40 mM, and more preferably about 20 mM. The DNA extraction composition comprises a buffer in the amount from about 1 mM to about 300 mM, preferably from about 10 mM to about 150 mM, more preferably about 50 mM. The pH of the buffer is from about 6.0 to about 8.8, preferably from about 7.5 to about 8.3, and more preferably about 7.8. Non-limiting examples of the buffer include acetate, BES, citrate, glycine, HEPES, MES, phosphate, PIPES, Tricine and Tris. It is preferred to use Tris. The DNA extraction composition may optionally comprise a non-ionic detergent from about 0.1% to about 50%, preferably from about 0.5% to about 10%, and more preferably about 1%. Non-limiting examples of non-ionic detergent include Nonidet NP-40, Triton(copyright) X-100 (octoxynol), Tween(copyright) 20 (polyoxyethylenesorbitan monolaurate) and Tween(copyright) 80 (polyoxyethylenesorbitan monooleate). It is preferred to use Tween(copyright) 80.
In accordance with a second aspect of the present invention, a simple, rapid, highthroughput method is provided to release DNA from a biological sample, especially from a biological sample adsorbed to a cellulosic material, such as cotton based papers. The method can be performed in a single tube, thus greatly simplifying the DNA extraction process for biological samples, especially blood, dried on cotton based papers. According to the present invention, the DNA extraction method comprises (a) contacting a biological sample with the DNA extraction composition described above, (b) incubating the mixture at a low temperature, (c) incubating the mixture at an elevated temperature and (d) isolating the supernatant which contains the solubilized, extracted DNA.
A disk of cellulosic material containing a biological material is added to a tube or a well, such as a microtiter well. Any mass of substrate can be immersed in this composition for DNA extraction. Commercial paper punches are available that produce sizes useful for 96-well format plates (1-5 mm). The process of the present invention performs well with a variety of punch sizes and with various numbers of punches extracted as a single sample. These conditions include sizes of the disks is in the range from about I mm to about 5 mm, with between 1 and 20 punches per well. The DNA extraction composition described herein is added to the tube or well containing the disk(s) in an amount from about 20 xcexcL to about 300 xcexcL, preferably from about 40 xcexcL to about 100 xcexcL, and more preferably about 50 xcexcL. The disk(s) and DNA extraction solution is first incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at a temperature of from about 40xc2x0 C. to about 60xc2x0 C., preferably from about 10xc2x0 C. to about 45xc2x0 C., and more preferably at room temperature (25xc2x0 C.). The disk(s) and DNA extraction solution is then incubated for about 0.5 minutes to about 60 minutes, preferably for about 5 minutes to about 20 minutes, and more preferably for about 10 minutes at an elevated temperature of from about 45xc2x0 C. to about 100xc2x0 C., preferably from about 55xc2x0 C. to about 100xc2x0 C., and more preferably at 95xc2x0 C. The supernatant is then isolated using any suitable technique. One suitable technique is centrifugation, such as 3000 xc3x97 g for 10 minutes. The supernatant can then be used in molecular techniques such as amplification reactions, hybridization analysis, sequencing and restriction analysis. It has been found that the supernatant can be used as target for amplification reactions following a dilution of 5-10 fold, preferably 8-fold, at a 10% to 20% final reaction volume.